Aspergillus fumigatus is a potentially virulent species of filamentous fungus, particularly in immunocompromised individuals. A. fumigatus reproduces by producing large numbers of airborne conidia which, once inhaled by susceptible patients, can lead to life-threatening invasive aspergillosis (1). Bone marrow and solid organ transplant recipients (2), cancer patients (3), AIDS patients (4), and those with chronic granulomatous disease (5) are particularly at risk for developing invasive aspergillosis. Invasive aspergillosis can be treated with the anti-fungal drugs amphotericin B and itraconazole; however, these drugs have low rates of success. Even with prophylaxis and treatment with amphotericin B, mortality rates average 65% for pulmonary aspergillosis and approach 100% if the disease spreads to the central nervous system (6). The basic virulence factors that allow some Aspergillus species to establish invasive infections remain unclear (1, 7). Approximately 90% of invasive aspergillosis cases are caused by one species, Aspergillus fumigatus. The remainder are caused primarily by Aspergillus nidulans, Aspergillus flavus, Aspergillus niger and Aspergillus terreus (8).
The number of patients undergoing solid-organ transplantation or hematopoietic stem cell transplant (HSCT) has increased rapidly in the last two decades. Worldwide, 15,000 allogeneic and 25,000 autologous stem cell transplants were performed (9). In the US, an average of 23,000 were performed annually between 1998-2002, a 20% increase over the preceding 5 year period (United Network of Organ Sharing). Invasive aspergillosis is now a leading cause of death in leukemia and bone marrow transplant units; Aspergillus infections have been reported in 2-26% of hemopoietic stem cell transplant patients, and 1-15% of organ transplant recipients. An estimated 9.3-16.9% of all deaths in transplant recipients in the first year are attributable to invasive aspergillosis (10). Because of the difficulty in diagnosing invasive aspergillosis, and the high mortality rate of the infection, new diagnostic and treatment methods are urgently needed.
As described below, the inventors have determined that the virulence of A. fumigatus appears to be related to the secretion of siderophores, such as N′,N″,N′″-triacetylfusarinine C (TAF) and ferricrocin. Siderophores are microbial iron chelates synthesized by some fungi and bacteria in response to low iron availability in the environment, such as serum. The inventors have discovered that TAF and ferricrocin appear to be able to remove iron from human serum iron-binding proteins, such as transferrin.
In light of this discovery, the need has arisen for methods for preventing or treating fungal infections caused by by A. fumigatus or other Aspergillus species by inhibiting secretion of siderophores. Since siderophore biosynthesis pathways are absent in human cells, the pathways present attractive new targets for antimicrobial chemotherapy. The first committed step in siderophore biosynthesis is catalyzed by the enzyme L-ornithine N5-oxygenase. A subsequent step in siderophore biosynthesis is catalyzed by non-ribosomal peptide synthetases. The invention therefore includes methods for identifying candidate drugs capable of inhibiting ornithine oxygenase, inhibiting other enzymes involved in siderophore biosynthesis such as non-ribosomal peptide synthetases, inhibiting the secretion of siderophores, or interfering with the formation of the siderophore-iron complex.
The need has also arisen for a diagnostic test using a biomarker indicative of likely A. fumigatus infection. Invasive aspergillosis is difficult to diagnose by radiological means. Radiological findings are often varied and non-specific, including segmental and multilobar consolidation, perihilar infiltrates, multiple small nodules, peripheral nodular masses and pleural effusions (11). Additionally, definitive diagnosis requires histopathologic evidence of deep tissue invasion or a positive culture from a sterile site. However, obtaining tissue from these thrombocytopenic patients is dangerous, and blood/CSF cultures are seldom positive. fumigatus can be cultured from bronchoalveolar lavage from infected patients. However, this is not a consistent finding, and often does not occur until late in infection. Genomic DNA can be detected in blood using PCR methods. Such methods are specific but are only semi-quantitative and are expensive. The simplest and cheapest method currently available is the PLATELIA™ Aspergillus ELISA test from Bio-Rad. This measures circulating galactomannan, a carbohydrate shed from the fungal cell wall. The specificity of the assay is very good (85%); however, the sensitivity of the assay is quite variable (29-100%). In addition, false positive reactions have been shown to occur, especially in children (83%) (12). Therefore new diagnostic methods are urgently needed.